Reduction of matrix metalloproteinase-9 expression by culture filtrate of Paecilomyces farinosus J3

Abstract

The aim of the present study was to investigate the anti-tumor effects of a culture filtrate of Paecilomyces farinosus J3. Various anti-tumor assays using B16 melanoma cells were carried out. Paecilomyces farinosus J3 significantly decreased the wound healing capability, invasiveness and angiogenic activity, which was confirmed by wound healing, human umbilical vein endothelial cell and invasion assays. Paecilomyces farinosus J3 strongly inhibited cell migration, tube formation and the angiogenic process in a concentration-dependent manner. Zymographic analysis also indicated a reduced expression of matrix metalloproteinase-9 (MMP-9), a 92-kDa gelatinase. Taken together, the results indicate that the anti-tumor activities of Paecilomyces farinosus J3 originate from the reduction of MMP-9 expression in B16F10 cells.

Figure 1.

Paecilomyces farinosus J3 inhibits cell migration in vitro. (A) Confluent monolayers of B16 cells were pre-treated with or without the Paecilomyces farinosus J3 culture filtrate. The monolayers were then wounded and stimulated with 100 ng PMA or left untreated (controls). The plates were photographed at 0 or 24 h post-wounding. Bar, 200 μm. (B) Quantification of the wound healing. Cell migration was quantified by counting the wound width 24 h after the plates were treated with or without PMA. Values are the means ± SD from five cultures each in duplicate experiments. CON, control. ^*Significant difference from the control, P<0.05. ^**Significant difference from PMA treatment alone, P<0.05.

Figure 2.

Paecilomyces farinosus J3 inhibits cell invasion in vitro. Cell invasion was assayed in a Matrigel-coated Transwell chamber. (A) Invasion was compared among B16 cells exposed to no treatment (control; a), 100 μg/ml of Paecilomyces farinosus J3 (b), 75 nM PMA (c) and both Paecilomyces farinosus J3 and PMA (d). Representative fields of migrated cells were photographed. Bar, 200 μm. (B) Quantification of the invasion activity. Cell migration was quantified 24 h after the cells were exposed to no treatment (control; CON), Paecilomyces farinosus J3, PMA, or both Paecilomyces farinosus J3 and PMA. Migrated cells were counted from five randomly selected microscopic fields and the results are given as the average per field ± SD of three independent experiments. ^*Significant difference from the control, P<0.05. ^**Significant difference from PMA treatment alone, P<0.05.

Figure 3.

Paecilomyces farinosus J3 alleviates HUVEC tube formation on Matrigel. (A) HUVECs were plated at 2x10⁴ cells/well in a Matrigel-coated 24-well plate, and then exposed to 0, 0.1, 0.3, 1 or 3 mg/ml of Paecilomyces farinosus J3 culture filtrate. After 24 h, the culture medium was removed and the cells were fixed with 10% formalin. The cell morphology infiltrated into the Matrigel (A), and the relative tubular numbers (B) of the formed tubes were calculated. Bar, 10 μm. The results are shown as the average per field ± SD of three independent experiments. CON, control. ^*Significant difference from the control (Paecilomyces farinosus J3, 0 mg/ml), P<0.05.

Figure 4.

Paecilomyces farinosus J3 inhibits MMP-9 expression. (A) Proteins with gelatinolytic activity were identified by electrophoresis in the presence of SDS on 10% polyacrylamide gels containing 0.1% (w/v) gelatin. In brief, cell culture medium was obtained from cultured B16F1 cells in DMEM and PMA (75 nM), with or without different concentrations of J3 for 24 h in 6-well culture dishes. The cultured medium was mixed with SDS-PAGE sample buffer in the absence of β-mercaptoethanol and DTT. Five microliters of sample were loaded onto a gel.